Probiotic compositions and uses thereof

ABSTRACT

The present invention relates to an isolated bacterial strain of Lactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited at Food Industry Research and Development Institute, Taiwan, under accession number BCRC 911035. The present invention also relates to a probiotic composition comprising an isolated bacterial strain of Lactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited at Food Industry Research and Development Institute, Taiwan, under accession number BCRC 911035, and optionally, one or more additional probiotic organisms that enhance the probiotic activity of the Lactiplantibacillus plantarum subsp. plantarum MFM 30-3. The present invention further relates to a method for preventing or treating chronic kidney disease in a subject in need thereof comprising: administering to said subject a pharmaceutically effective amount of the probiotic composition comprising an isolated bacterial strain of Lactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited at Food Industry Research and Development Institute, Taiwan, under accession number BCRC 911035, and optionally, one or more additional probiotic organisms that enhance the probiotic activity of the Lactiplantibacillus plantarum subsp. plantarum MFM 30-3.

This application contains a Sequence Listing in a computer readableform. The computer readable form is incorporated herein by reference.The present application claims priority to TW Appl. No. 110104506, filedFeb. 5, 2021, incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a composition comprisingLactiplantibacillus plantarum subsp. plantarum MFM 30-3 (deposited theDSMZ Accession No. DSM 34213) either alone or in combination with one ormore probiotic organisms, or an agent that may enhance the probioticactivity of Lactiplantibacillus plantarum subsp. plantarum MFM 30-3.Further, the present invention provides administration ofLactiplantibacillus plantarum subsp. plantarum MFM 30-3 in the probioticcomposition to a subject in need for preventing or treating chronickidney disease.

BACKGROUND OF THE INVENTION

Chronic kidney disease (CKD) is characterized by a substantial loss ofkidney function and is emerging as a major risk factor forcardiovascular diseases. Thus, CKD is becoming a severe public healthissue that desperately requires a better solution to ameliorate andalleviate the progression of the disease. CKD symptoms are diverse andmainly stem from organic waste products, called uremic retention solutes(URSs), that are normally cleared by the kidneys and accumulate in CKDpatients. Among the URSs, protein-bound uremic toxins, such as indoxylsulfate (IS) and p-cresyl sulfate (PCS), are derived from microbialmetabolism and have deleterious effects on the cardiovascular system.Several treatments targeting URSs have been proposed, such as applyingoral adsorbents to decrease their absorption and performinghemodialysis/kidney transplantation to increase their clearance.However, most of these treatments have limitations and disadvantages(Davenport, A. More frequent hemodialysis does not effectively clearprotein-bound azotemic solutes derived from gut microbiome metabolism.Kidney Int. 2017, 91, 1008-1010).

Since the gut microbiota has a significant role in URSs production,numerous studies have revealed that the gut microbiota is associatedwith several pathological conditions, leading to accelerated CKDprogression. Iatrogenic effects and alterations of physiologicalconditions, including pharmacological therapies, a slow intestinaltransit time, impaired protein assimilation, and dietary restriction inCKD patients become the driving force to dysregulate the gut compositionand expand the imbalance between symbionts and pathobionts that favorspathobiont overgrowth. However, the causality between the progression ofCKD and gut dysbiosis is not completely understood.

Strategies based on microbiota-based therapeutic interventions, with theaim of modulating the gut microbiota and restoring the gut homeostasisby increasing the symbiotic bacteria or by absorptive removal ordegradation of gut-derived precursors, could be considered as effectiveapproaches to reduce uremic toxins. The efficacy of probiotics indecreasing uremic toxin production and improving renal functions hasbeen investigated in in vitro models and in various animal and human CKDstudies. However, to date, in vitro screening platforms with qualityintervention trials examining this novel CKD therapy are still lacking.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Indole and p-cresol clearance ability of 10⁹ CFU/mL of teststrains (S1: MFM 22; S2: MFM 30-2; S3: MFM 30-3; S4: MFM 18; S5: MFM14-1; D1: MFM 22/18; D2: MFM 30-2/18; D3: MFM 30-3/18; T1: MFM22/18/14-1; T2: MFM 30-2/18/14-1; T3: MFM 30-3/18-14-1) after 48 h ofincubation in simulated intestinal juice. The results are presented asmean±SEM (n=3). Means in columns with different letters aresignificantly different (p<0.05).

FIG. 2A: Representative images of H&E- and MT-stained kidney sections.Scale bar=100 μm. The kidney injury score and quantitative analysis ofthe interstitial fibrosis area are determined based on the H&E and MTresults, respectively. The results are presented as mean±SEM (n=9-11).The symbols indicate a significant difference compared with the control(*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 2B: Western blot analysis of kidney fibrosis-related proteins arenormalized to the beta-actin levels. The results are presented asmean±SEM (n=9-11). The symbols indicate a significant differencecompared with the control (*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 2C: BUN and CRE analyses. The results are presented as mean±SEM(n=9-11). The symbols indicate a significant difference compared withthe control (*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 3A: Activity of antioxidative protein. The results are presented asmean±SEM (n=9-11). The symbols indicate a significant differencecompared with the control (*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 3B: Concentration of cytokines. The results are presented asmean±SEM (n=9-11). The symbols indicate a significant differencecompared with the control (*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 3C: Western blot analysis of kidney oxidative stress andinflammatory response indicators are normalized to the beta-actin level.The results are presented as mean±SEM (n=9-11). The symbols indicate asignificant difference compared with the control (*p<0.05) and CKDgroups (^(#)p<0.05).

FIG. 4: Colonic uremic toxin precursors and serum uremic toxins. (A)Concentration of uremic toxin precursors in the colonic content. (B)Concentration of uremic toxins in serum. The results are presented asmean±SEM (n=9-11). The symbols indicate a significant differencecompared with the control (*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 5A: Serum concentration of FITC-dextran. The results are presentedas mean±SEM (n=9-11). The symbols indicate a significant differencecompared with the control (*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 5B: Fecal concentration of SCFA. The results are presented asmean±SEM (n=9-11). The symbols indicate a significant differencecompared with the control (*p<0.05) and CKD groups (^(#)p<0.05).

FIG. 6A: Comparison of alpha diversity between the colonic microbiome ofeach group (n=9-11). The symbols indicate a significant differencecompared with control (*p<0.05) and CKD mice (^(#)p<0.05).

FIG. 6B: PCA plot based on the relative abundance of bacterial taxa ofindividual mice.

FIG. 7A: Relative abundance of bacterial genera or species enriched inLD mice compared to CKD mice (n=9-11). The symbols indicate asignificant difference compared with the control (*p<0.05) and CKDgroups (^(#)p<0.05).

FIG. 7B: Relative abundance of bacterial genera or species enriched inLD mice compared to CKD mice (n=9-11). The symbols indicate asignificant difference compared with the control (*p<0.05) and CKDgroups (^(#)p<0.05).

FIG. 7C: Relative abundance of bacterial genera or species enriched inLD mice compared to CKD mice (n=9-11). The symbols indicate asignificant difference compared with the control (*p<0.05) and CKDgroups (^(#)p<0.05).

SUMMARY OF THE INVENTION

The present invention relates to an isolated bacterial strain ofLactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited underthe DSMZ Accession No. DSM 34213. The present invention also relates toa probiotic composition comprising an isolated bacterial strain ofLactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited underthe DSMZ Accession No. DSM 34213, and optionally, one or more additionalprobiotic organisms that enhance the probiotic activity of theLactiplantibacillus plantarum subsp. plantarum MFM 30-3. The presentinvention further relates to a method for preventing or treating chronickidney disease in a subject in need thereof comprising: administering tosaid subject a pharmaceutically effective amount of the probioticcomposition comprising an isolated bacterial strain ofLactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited underthe DSMZ Accession No. DSM 34213, and optionally, one or more additionalprobiotic organisms that enhance the probiotic activity of theLactiplantibacillus plantarum subsp. plantarum MFM 30-3.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, a novel in vitro screening platform isdeveloped to select potential probiotics with higher uremic toxinprecursor reducing properties. An adenine-induced CKD mouse model isfurther utilized to elucidate the functional properties of the selectedstrains on CKD progression. The mechanisms underlyingprobiotic-prevented CKD progression are also evaluated through analysisof the microbiota and metabolome.

After an in vitro screening assay and simulated gastric and intestinalfluid tests, two strains, Lactiplantibacillus plantarum subsp. plantarumMFM 30-3 and Lacticaseibacillus paracasei subsp. paracasei MFM 18, arechosen for further characterization. A combination of the above twostrains is selected and named lactic acid bacteria mix (Lm) for furtherstudies. The results show that Lm significantly improved the kidneyfunction by reducing kidney injury and fibrotic-related proteins.Furthermore, Lm decreases oxidative stress and proinflammatory reactionsand elevates immune responses in the kidney. Importantly, Lm reversesgut dysbiosis and restores the abundance of commensal bacteria,especially short-chain fatty acid producers, leading to improvedintestinal barrier integrity via modulation of microbial composition andmetabolite production. Taken together, these findings provide evidencethat Lm can be a preventive or even therapeutic approach against CKD.

The term “Lactiplantibacillus plantarum subsp. plantarum” is formerlyknown as “Lactobacillus plantarum subsp. plantarum” which has beenofficially reclassified from April 2020 according to Zheng J et al.(Zheng J, Wittouck S. et al., (2020) ‘A taxonmonic note on the genusLactobacillus: Description of 23 novel genera, emended description ofthe genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceaeand Leuconostocaceae’. Int. J. Syst. Evol. Microbiol, 70(4): 2782-2858).

The term “Lacticaseibacillus paracasei subsp. paracasei” is formerlyknown as “Lactobacillus paracasei subsp. paracasei” which has also beenofficially reclassified from April 2020 according to Zheng J et al.(Zheng J, Wittouck S. et al., (2020) ‘A taxonmonic note on the genusLactobacillus: Description of 23 novel genera, emended description ofthe genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceaeand Leuconostocaceae’. Int. J. Syst. Evol. Microbiol, 70(4): 2782-2858).

The term “probiotic” is intended to mean a microorganism (such as abacterium or a yeast) having a beneficial effect on the general healthof an animal or a human, or a beneficial effect on a specific healthproblem, disorder or disease, alleviating pain, symptoms or discomfortassociated with those health problem, disorder or disease.

The expression “probiotic composition” is intended to mean a generalproduct comprising at least one probiotic. It will be understood thatthis expression encompasses a variety of specific products, allpresenting the characteristic of comprising at least one probiotic.

The term “CFU”, or “colony forming unit”, is a unit commonly used toestimate the concentration of microorganisms in a test sample. Thenumber of visible colonies (CFU) present on an agar plate can bemultiplied by the dilution factor to provide a CFU/ml result.

The term “intestinal barrier”, also referred to as “intestinal mucosalbarrier”, refers to the property of the intestinal mucosa that ensuresadequate containment of undesirable luminal contents within theintestine while preserving the ability to absorb nutrients. Theseparation it provides between the body and the gut prevents theuncontrolled translocation of luminal contents into the body. Its rolein protecting the mucosal tissues and circulatory system from exposureto pro-inflammatory molecules, such as microorganisms, toxins, andantigens is vital for the maintenance of health and well-being.

The term “about” as used herein is intended to reflect a variation of10% of the value it is attached to. For example, a concentration of“about 20%” is reflective of a concentration ranging from 18% to 22%. Asanother example, a quantity of “about 10⁸” is reflective of a range of0.9×10⁸ to 1.1×10⁸.

Therefore, the present invention provides an isolated bacterial strainof Lactiplantibacillus plantarum subsp. plantarum MFM 30-3 depositedunder the Budapest Treaty at Leibniz Institute DSMZ-German Collection ofMicroorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig,Germany) on Mar. 22, 2022 and has been given the DSMZ Accession No. DSM34213 by the International Depositary Authority. The present inventionalso provides an isolated bacterial strain of Lacticaseibacillusparacasei subsp. paracasei MFM 18 deposited under the Budapest Treaty atLeibniz Institute DSMZ-German Collection of Microorganisms and CellCultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on Mar. 22,2022 and has been given the DSMZ Accession No. DSM 34212 by theInternational Depositary_Authority. The deposits were made for a term ofat least thirty (30) years and at least five (5) years after the mostrecent request for the furnishing of a sample of the deposit wasreceived by the depository. All restrictions imposed by the depositor onthe_availability to the public of the deposited materials will beirrevocably removed upon the granting of the patent.

The present invention further provides a probiotic compositioncomprising an isolated bacterial strain of Lactiplantibacillus plantarumsubsp. plantarum MFM 30-3 deposited under the DSMZ Accession No. DSM34213, and optionally, one or more additional probiotic organisms thatenhance the probiotic activity of the Lactiplantibacillus plantarumsubsp. plantarum MFM 30-3. Also, a probiotic composition comprising anisolated bacterial strain of Lacticaseibacillus paracasei subsp.paracasei MFM 18 deposited under the DSMZ Accession No. DSM 34212, andoptionally, one or more additional probiotic organisms that enhance theprobiotic activity of the Lacticaseibacillus paracasei subsp. paracaseiMFM 18 is provided.

In an embodiment, the probiotic composition of the present inventioncomprises an isolated bacterial strain of Lactiplantibacillus plantarumsubsp. plantarum MFM 30-3 deposited under the DSMZ Accession No. DSM34213, and an isolated bacterial strain of Lacticaseibacillus paracaseisubsp. paracasei MFM 18 deposited under the DSMZ Accession No. DSM34212. In another embodiment, the quantity ratio of MFM 30-3:MFM 18 inthe probiotic composition of the present invention is between 2:1 and1:2. In yet another embodiment, the quantity ratio of MFM 30-3:MFM 18 inthe probiotic composition of the present invention is 1:1.

The probiotic composition of the present invention may be presented insolid, liquid or semi solid form and may be taken by routes selectedfrom oral, rectal, or parenteral. The probiotic composition according tothe invention may additionally comprises one or more suitablepharmaceutical/neutraceutical excipients/carriers to provide the same ina desired dosage form to achieve desired delivery. The suitablepharmaceutical/neutraceutical excipients may be selected from the groupconsisting of diluents, binders, polymers, fillers, vehicles, carriers,and disintegrants.

The probiotic composition of the present invention is suitable for useas a food, a food supplement, a nutraceutical or as a therapeutic.

The present invention still provides a method for preventing or treatingchronic kidney disease in a subject in need thereof comprising:administering to said subject a pharmaceutically effective amount of aprobiotic composition comprising an isolated bacterial strain ofLactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited underthe DSMZ Accession No. DSM 34213, and optionally, one or more additionalprobiotic organisms that enhance the probiotic activity of theLactiplantibacillus plantarum subsp. plantarum MFM 30-3. In anembodiment, the probiotic composition comprises an isolated bacterialstrain of Lactiplantibacillus plantarum subsp. plantarum MFM 30-3deposited under the DSMZ Accession No. DSM 34213, and an isolatedbacterial strain of Lacticaseibacillus paracasei subsp. paracasei MFM 18deposited under the DSMZ Accession No. DSM 34212. In an embodiment, thedosage of the probiotic composition is about 5×10⁵ to about 5×10⁹CFU/ml. In another embodiment, the dosage of the probiotic compositionis at least 5×10⁶ CFU/ml. In yet another embodiment, the dosage of theprobiotic composition is at least 5×10⁷ CFU/ml. In an embodiment, thequantity ratio of MFM 30-3:MFM 18 in the probiotic composition used inthe method is between 2:1 and 1:2. In another embodiment, the quantityratio of MFM 30-3:MFM 18 in the probiotic composition used in the methodis 1:1.

The present invention also provides a method for preventing or treatingchronic kidney disease in a subject in need thereof comprising:administering to said subject a pharmaceutically effective amount of aprobiotic composition comprising an isolated bacterial strain ofLacticaseibacillus paracasei subsp. paracasei MFM 18 deposited under theDSMZ Accession No. DSM 34212, and optionally, one or more additionalprobiotic organisms that enhance the probiotic activity of theLacticaseibacillus paracasei subsp. paracasei MFM 18. In an embodiment,the probiotic composition comprises an isolated bacterial strain ofLactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited underthe DSMZ Accession No. DSM 34213, and an isolated bacterial strain ofLacticaseibacillus paracasei subsp. paracasei MFM 18 deposited under theDSMZ Accession No. DSM 34212. In an embodiment, the dosage of theprobiotic composition is about 5×10⁵ to about 5×10⁹ CFU/ml. In anotherembodiment, the dosage of the probiotic composition is at least 5×10⁶CFU/ml. In yet another embodiment, the dosage of the probioticcomposition is at least 5×10⁷ CFU/ml. In an embodiment, the quantityratio of MFM 30-3:MFM 18 in the probiotic composition used in the methodis between 2:1 and 1:2. In another embodiment, the quantity ratio of MFM30-3:MFM 18 in the probiotic composition used in the method is 1:1.

In an embodiment, the method for preventing or treating chronic kidneydisease of the present invention reduces the content of the indicativemolecule selected from indole, p-cresol, indoxyl sulfate, or p-cresylsulfate. In another embodiment, the method for preventing or treatingchronic kidney disease of the present invention reverses gut dysbiosisand restores the abundance of commensal bacteria. In yet anotherembodiment, the method for preventing or treating chronic kidney diseaseof the present invention improves intestinal barrier integrity viamodulation of microbial composition and metabolite production.

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

Example 1 Materials and Methods Bacterial Strains

Lactic acid bacteria (LAB) strains were isolated from Mongolianfermented milk (MFM). For strain isolation, the process of Watanabe etal. (Watanabe, K.; Fujimoto, J.; Sasamoto, M.; Dugersuren, J.; Tumursuh,T.; Demberel, S. Diversity of lactic acid bacteria and yeasts in Airagand Tarag, traditional fermented milk products World J. Microbiol.Biotechnol. 2008, 24, 1313-1325) was adopted with slight modifications.Briefly, homogenized samples were subjected to serial 10-fold dilutionswith saline and 0.1 mL aliquots were inoculated onto modified MRS agarplates supplemented with 2% lactose (Bioshop Canada Inc, Canada), 0.001%both of cycloheximide (Sigma-Aldrich), and sodium azide (Sigma-Aldrich)and incubated anaerobically at 30° C. for 3 days. The colonies wereselected according to distinct morphologies (size, color, and shape).The isolates were then purified by streaking at least three times on themodified MRS agar plates. For strain discrimination of the isolates,Enterobacterial repetitive intergenic consensus polymerase chainreaction (ERIC-PCR) analysis was performed as previously described(Ventura, M.; Meylan, V.; Zink, R. Identification and tracing ofBifidobacterium species by use of enterobacterial repetitive intergenicconsensus sequences. Appl. Environ. Microbiol. 2003, 69, 4296-4301). Onthe basis of the resulting ERIC-PCR profiles, one representative strainfor each of the 40 groups were chosen and cultured for frozen storageand further analysis. Followed by the detailed strain typing of the 40strains by the combination of ERIC-PCR and random amplified polymorphicDNA (RAPD)-PCR, the 20 strains were discriminated from 12 strains, whichwere assigned with branch numbers (as listed in Table 1). Beforesubsequent analysis, the isolates were cultured twice with 1% inoculumin modified MRS broth (2% lactose) at 30 or 37° C. for 24 or 48 hdepending on different strains.

TABLE 1 Indole Clearance Ability of LAB Strains indole clearance ability(%) after hrs of incubation strain 0 24 48 72 MFM 1 3.81 ± 0.43 4.57 ±0.19 2.85 ± 1.03 6.57 ± 0.67 MFM 2 2.28 ± 0.54 4.32 ± 0.33 4.02 ± 0.795.35 ± 0.98 MFM 2-1 1.95 ± 0.27 4.15 ± 0.19 4.47 ± 1.33 5.30 ± 0.57 MFM2-2 3.81 ± 0.31 3.36 ± 0.25 4.55 ± 1.39 4.09 ± 0.82 MFM 2-3 4.61 ± 0.564.37 ± 0.35 5.01 ± 0.51 4.65 ± 0.64 MFM 3 4.48 ± 0.84 4.77 ± 0.88 5.35 ±0.15 5.90 ± 0.51 MFM 3-1 4.04 ± 0.49 4.46 ± 0.14 5.59 ± 0.82 6.04 ± 0.63MFM 3-2 4.04 ± 0.71 3.95 ± 0.54 4.94 ± 0.36 4.80 ± 1.32 MFM 3-3 4.24 ±0.27 4.01 ± 0.14 4.74 ± 0.55 5.01 ± 0.66 MFM 4 2.86 ± 0.60 3.24 ± 0.515.14 ± 1.21 3.96 ± 0.12 MFM 5 2.34 ± 0.38 4.01 ± 0.73 4.28 ± 0.57 2.67 ±1.30 MFM 6 2.02 ± 0.39 1.88 ± 0.60 4.22 ± 0.22 3.87 ± 1.29 MFM 7 1.33 ±0.35 2.25 ± 0.26 5.54 ± 1.90 4.23 ± 0.56 MFM 8 3.90 ± 1.22 3.16 ± 0.766.28 ± 0.77 6.17 ± 0.41 MFM 9 2.01 ± 0.36 4.79 ± 0.67 3.08 ± 0.72 2.02 ±0.27 MFM 10 2.75 ± 0.45 5.53 ± 0.23 4.95 ± 0.74 5.68 ± 0.96 MFM 11 3.60± 0.19 5.75 ± 1.92 6.71 ± 0.31 6.72 ± 1.21 MFM 12 −4.97 ± 1.94  3.01 ±0.43 5.55 ± 0.13 7.33 ± 0.47 MFM 13 4.78 ± 0.16 4.79 ± 0.43 5.37 ± 0.495.53 ± 0.16 MFM 13-1 2.86 ± 0.30 3.88 ± 0.50 3.92 ± 1.20 4.14 ± 1.48 MFM13-2 4.16 ± 0.54 4.70 ± 0.72 3.64 ± 0.62 3.83 ± 0.79 MFM 14-1 −4.46 ±6.75  2.57 ± 0.72 5.87 ± 0.14 8.50 ± 1.96 MFM 14-2 −5.93 ± 2.71  2.73 ±0.21 6.40 ± 2.30 6.59 ± 0.47 MFM 15-1 3.86 ± 0.10 4.15 ± 0.54 2.54 ±0.36 5.91 ± 0.20 MFM 15-1-1 3.94 ± 0.09 4.85 ± 0.20 1.98 ± 0.49 6.20 ±0.20 MFM 15-1-2 3.30 ± 0.79 4.61 ± 0.90 6.06 ± 0.56 5.77 ± 0.57 MFM 15-23.89 ± 0.20 5.00 ± 0.28 5.38 ± 1.05 6.22 ± 1.25 MFM 16 4.32 ± 0.64 4.87± 0.98 4.55 ± 1.38 3.54 ± 2.17 MFM 17 2.84 ± 0.37 6.25 ± 0.27 4.37 ±0.65 5.40 ± 0.63 MFM 18 4.82 ± 0.08 6.57 ± 0.09 9.22 ± 1.14 10.13 ±0.72  MFM 19 3.01 ± 0.58 3.25 ± 0.54 2.99 ± 0.86 2.76 ± 1.04

Uremic Toxin Precursor Clearance Ability of LAB Strains

Before test, the 60 isolates were cultured twice with 1% inoculum inmodified MRS broth (2% lactose) at 30 or 37° C. for 24 or 48 h dependingon different strains. All isolates reached the stationary phase withcell numbers ranging from 10⁹ to 10¹⁰ CFU/mL. Cells of test strains werecentrifuged at 3300×g for 10 min. After discarding the supernatant, thecells were incubated in simulated intestinal juice (0.05 M KH₂PO₄, pH7.25) with 200 ppm indole or 100 ppm p-cresol for 24-72 h. Afterincubation, the cells were centrifuged again, the supernatant wascollected, and the concentrations of indole and p-cresol weredetermined. The potential strains with high clearance ability wereselected. To choose the possible combination with the best clearanceresult, the selected strains were adjusted to 10⁹ CFU/mL with saline andperformed the uremic toxin precursor clearance test, as describedpreviously.

Identification of LAB

Genomic DNA of the LAB strains was extracted. The 16S rRNA gene wasamplified with the primers 8F and 15R (Watanabe, K.; Fujimoto, J.;Sasamoto, M.; Dugersuren, J.; Tumursuh, T.; Demberel, S. Diversity oflactic acid bacteria and yeasts in Airag and Tarag, traditionalfermented milk products of Mongolia. World J. Microbiol. Biotechnol.2008, 24, 1313-1325). Phenylalanyl-tRNA synthase (pheS) and RNApolymerase A subunit (rpoA) genes were amplified with the primerspheS-21-F and pheS-23-R and rpoA-21-F and rpoA-23-R, respectively(Naser, S. M.; Thompson, F. L.; Hoste, B.; Gevers, D.; Dawyndt, P.;Vancanneyt, M.; Swings, J. Application of multilocus sequence analysis(MLSA) for rapid identification of Enterococcus species based on rpoAand pheS genes. Microbiology 2005, 151, 2141-2150). Full-lengthsequencing of the 16S rRNA gene was performed with the primers 350F,520R, and 930F (Miyake, T.; Watanabe, K.; Watanabe, T.; Oyaizu, H.Phylogenetic analysis of the genus Bifidobacterium and related generabased on 16S rDNA sequences. Microbiol. Immunol. 1998, 42, 661-667), andpartial sequencing of the pheS and rpoA genes was performed with thepreviously described primers. All sequence analyses were carried out atGenomics BioSci & Tech Co., Ltd. (New Taipei, Taiwan). The sequenceswere assembled by using Chromas version 2.23 (Technelysium Pty. Ltd.,QLD, Australia), GENETYX version 5.1, and GENETYX ATSQ version 1.03(Software Development Co., Tokyo, Japan). Phylogenetic trees wereconstructed by the neighbor-joining method by using Clustal X softwareversion 2.1 (Thompson, J.; Gibson, T. J.; Plewniak, F.; Jeanmougin, F.;Higgins, D. G. The CLUSTAL_X windows interface: flexible strategies formultiple sequence alignment aided by quality analysis tools. NucleicAcids Res. 1997, 25, 4876-4882). The statistical reliability of thetrees was evaluated by bootstrap analysis of 1000 replicates by usingMEGA7 v7.0.14 software according to Kimura's two-parameter model as asubstitution model (Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecularevolutionary genetics analysis version 7.0 for bigger datasets. Mol.Biol. Evol. 2016, 33, 1870-1874).

Gastrointestinal Tolerance

LAB isolated strains were cultured twice with 1% inoculum in modifiedMRS broth (2% lactose) at 30 or 37° C. for 24 h depending on differentstrains. All isolates reached the stationary phase with cell numbersranging from 10⁹ to 10¹⁰ CFU/mL. Cells of test strains were centrifugedat 3300×g for 10 min and washed twice with saline. After discarding thesupernatant, cells were completely dispersed in simulated gastric juice(3.6 mM CaCl₂, 1.5 mM MgCl₂, 49 mM NaCl, 12 mM KCl, and 6.4 mM KH₂PO₄,pH 2.0) containing 1600 U/mL pepsin (Sigma-Aldrich) and incubated at 37°C. with 120 rpm shaking for 1 h. Then, the cells were dispersed insimulated intestinal juice (0.1 M NaHCO₃, pH 8.0) containing 4.4 g/Lporcine bile and 1 g/L pancreatin (Sigma-Aldrich) for 2 h. Survivingbacteria were counted in modified MRS agar aerobically incubated at 30and 37° C. for 2 days (Yonekura, L.; Sun, H.; Soukoulis, C.; Fisk, I.Microencapsulation of Lactobacillus acidophilus NCIMB 701748 in matricescontaining soluble fibre by spray drying: Technologicalcharacterization, storage stability and survival after in vitrodigestion. J. Funct. Foods 2014, 6, 205-214).

Adenine-Induced CKD Animal Model

Male C57BL/6 mice (5 weeks old) were purchased from the NationalLaboratory Animal Center (Taipei, Taiwan) and housed in a specificpathogen-free (SPF) facility in the National Taiwan University AnimalResource Center with a 12 h light/dark cycle and free access tosterilized AIN93G pellets (Research Diets, Inc., New Brunswick, NJ, USA)and water. At 7 weeks of age, the mice were randomly divided into fourgroups [control, CKD, low dosage (LD), and high dosage (HD)]. The LD andHD groups were orally gavaged with 200 μL of a cell suspension equal tothe low dosage [10⁷ colony forming units (CFUs)/mice/day] and highdosage (10⁹ CFUs/mice/day) of mixed lactic acid strains for 6 weeks,respectively. At the third week, all groups except for the control groupwere fed an AIN93G diet supplemented with 0.2% adenine (Research Diets)to induce CKD for 18 days and then returned to their original feed. Atthe end of the animal study, the mice were euthanized, and blood,organs, feces, and the colonic content were collected for furtheranalysis. All of the animal experiments were approved by theInstitutional Animal Care and Use Committee of National TaiwanUniversity (IACUC approval no: NTU-107-EL-00053).

Biochemical Measurements

The assays for blood urea nitrogen (BUN) and creatinine (CRE) in serumwere analyzed by an automated clinical chemistry analyzer (FujifilmCorporation, Tokyo, Japan).

Indole and p-Cresol Analyses

In vitro, the concentrations of indole and p-cresol in the clearanceability test were measured using a high-performance liquidchromatography (HPLC) system (Jasco International Co. Ltd., Tokyo,Japan) and a Reprosil 100 C18 column (250×4.6 mm; 5 μm particle size;Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) with an injection volumeof 20 μL and a 1.0 mL/min flow rate. For analysis of the indoleconcentration, the mobile phase consisted of methanol and ultrapurewater (65:35), and the wavelength was set at 254 nm. For analysis of thep-cresol concentration, the mobile phase consisted of methanol, ACN, andultrapure water (15:26.3:58.7), and the wavelength was set to 210 nm. Invivo, due to the limited amounts of colonic contents and lowconcentrations of both precursors in colonic samples, a highersensitivity and efficiency approach was adapted from Liu et al. (Liu,Y.; Li, J.; Yu, J.; Wang, Y.; Lu, J.; Shang, E. X.; Zhu, Z.; Guo, J.;Duan, J. Disorder of gut amino acids metabolism during CKD progressionis related with gut microbiota dysbiosis and metagenome change. J.Pharm. Biomed. Anal. 2018, 149, 425-435). The colonic contents werehomogenized in PBS (FastPrep-24 5G Instrument, MP Biomedicals, Irvine,Calif., USA) and centrifuged at 16,100×g for 10 min at 4° C., and thesupernatant was mixed with ACN (1:3, v/v). Analytes were measured usinga HPLC system (Jasco International Co. Ltd., Tokyo, Japan) and aReprosil 100 C18 column (250×4.6 mm; 5 μm particle size; Dr. MaischGmbH, Ammerbuch-Entringen, Germany). Mobile phase A was 200 mM ammoniumformate (pH 4.5), and mobile phase B was 100% ACN. The analyticalconditions were isocratic at 48% B, and indole and p-cresol were elutedat 10.3 and 6.7 min, respectively. The injection volume was 20 μL, theflow rate was 1 mL/min, the autosampler tray temperature was 4° C., andthe fluorescence detection settings for indole were λ_(ex) 270 nm/λ_(em)340 nm and for p-cresol were λ_(ex) 260 nm/λ_(em) 300 nm.

Uremic Toxin Analysis

Serum was mixed with ACN (1:3, v/v), vortexed for 30 s, and thencentrifuged at 9,300×g for 5 min at room temperature. Serum uremic toxinwas measured using an HPLC system and a Reprosil 100 C18 column. Mobilephase A was 50 mM ammonium formate (pH 4.5), and mobile phase B was 100%ACN. The optimized conditions of the eluting gradient were as follows:5-40% B (0-13.0 min), 40% B (13.0-16.0 min), and 5% B (16.0-22.0 min).The injection volume was 20 μL, the flow rate was 1 mL/min, and theautosampler tray temperature was 4° C. Indoxyl sulfate and p-cresylsulfate were eluted at 11.0 and 12.6 min, respectively. The fluorescencedetection settings for indoxyl sulfate were λ_(ex) 300 nm/λ_(em) 390 nmand for p-cresyl sulfate were λ_(ex) 260 nm/λ_(em) 283 nm (Pretorius, C.J.; McWhinney, B. C.; Sipinkoski, B.; Johnson, L. A.; Rossi, M.;Campbell, K. L.; Ungerer, J. P. J. Reference ranges and biologicalvariation of free and total serum indoxyl- and p-cresyl sulphatemeasured with a rapid UPLC fluorescence detection method. Clin. Chim.Acta 2013, 419, 122-126).

Short-Chain Fatty Acid Analysis

The concentrations of fecal short-chain fatty acids (SCFAs) weremeasured as previously described with slight modifications (Torii, T.;Kanemitsu, K.; Wada, T.; Itoh, S.; Kinugawa, K.; Hagiwara, A.Measurement of short-chain fatty acids in human faeces usinghigh-performance liquid chromatography: specimen stability. Ann. Clin.Biochem. 2010, 47, 447-452). After a series of procedures, the obtainedfatty acids were dissolved in 200 μL of methanol. The concentration ofSCFAs was measured using a HPLC system with a Reprosil 100 C18 column.The mobile phase consisted of ACN, methanol, and ultrapure water(30:16:54), and the pH was adjusted to 4.5 with 0.1% TFA(Sigma-Aldrich). The injection volume was 30 μL, the flow rate was 1.1mL/min, the column temperature was 50° C., and the wavelength was set at400 nm.

Kidney Antioxidant Enzyme Activity Analysis

The levels of glutathione (GSH), superoxide dismutase (SOD), catalase,and glutathione peroxidase (GPx) in the kidney were determined using acommercial kit (Cayman Chemical Co., Ann Arbor, Mich., USA) according tothe manufacturer's instructions.

Kidney Cytokine Analysis

The tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 levelsin plasma were measured using ELISA kits (R&D system, Minneapolis,Minn., USA) according to the manufacturer's instructions.

Intestinal Permeability

Fluorescein isothiocyanate (FITC)-dextran (FD4) (Sigma-Aldrich) wasdissolved in PBS (60 mg/mL), and mice were orally gavaged with 200 μL ofFITC-dextran 4 h before blood collection. The serum was diluted 1:9 inPBS, and the fluorescence of FITC-dextran was determined using afluorometer with settings of λ_(ex) 485 nm/λ_(em) 528 nm (BioTek,Winooski, Vt., USA). Intestinal permeability was presented as theconcentration of serum FITC-dextran (Woting, A.; Blaut, M. Smallintestinal permeability and gut-transit time determined with low andhigh molecular weight fluorescein isothiocyanate-dextrans in C3H mice.Nutrients 2018, 10, 685).

Western Blot Analysis

Kidney lysates were separated by SDS-PAGE, transferred onto PVDFmembranes (Merck Millipore Ltd., Burlington, Mass., USA), and blotted at4° C. overnight with primary antibodies against transforming growthfactor (TGF)-β, fibronectin, collagen 1, myeloperoxidase (MPO),Toll-like receptor 4 (TLR4), and beta actin at a 1:1000 dilution, and atroom temperature for 1 h with an horseradish peroxidase-conjugated goatanti-rabbit IgG antibody at a 1:10,000 dilution (all from Abcam,Cambridge UK) as the secondary antibody. The membrane was incubated withWestern Lightning ECL Pro (PerkinElmer, Inc., Waltham, Mass., USA) anddetected using a ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

16S rRNA Gene Amplicon Sequencing

DNA was extracted from colonic contents. The V3-V4 regions of the 16SrRNA gene were amplified by the universal primers 341F(5′-CCTACGGGAGGCAGCAG-3′) (SEQ ID NO: 1) and 806R(5′-GGACTACCAGGGTATCTAAT-3′) (SEQ ID NO: 2) with barcodes and sequencedusing the Illumina MiSeq paired-end sequencing platform. Taxonomicannotation of the representative sequence for each operational taxonomicunit (OTU) was performed using the Ribosomal Database Project (RDP)classifier v2.2 against the Silva v.132 database. Alpha diversity(observed OTUs, Chao1, and Shannon) and beta diversity (principalcomponent analysis, PCA) were analyzed using QIIME v1.7.0 and R v2.15.3software. The linear discriminant analysis (LDA) effect size (LEfSe)algorithm was used for biomarker discovery to identify differentialenrichment of abundant taxa between groups. For functional analysis,functional abundances from 16S rRNA sequencing data were analyzed forthe prediction of functional genes with Phylogenetic Investigation ofCommunities by Reconstruction of Unobserved States (PICRUSt) v1.1.1 byusing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ofreference genomes (Langille, M. G. I.; Zaneveld, J.; Caporaso, J. G.;McDonald, D.; Knights, D.; Reyes, J. A.; Clemente, J. C.; Burkepile, D.E.; Vega Thurber, R. L.; Knight, R.; Beiko, R. G.; Huttenhower, C.Predictive functional profiling of microbial communities using 16S rRNAmarker gene sequences. Nat. Biotechnol. 2013, 31, 814-821).

Statistical Analysis

For in vitro studies, data were compared using one-way ANOVA withDuncan's test as a post hoc test. For in vivo studies, unpaired t-testsand nonparametric Mann-Whitney U tests were performed for all thephenotypic and next-generation sequencing (NGS) data. A P value of <0.05was considered statistically significant. Correlation analyses wereperformed by Spearman's correlation analysis. All statistical analyseswere performed by SPSS Statistics 23.0 (IBM, New York, N.Y., USA), SASv9.4 (SAS Institute Inc., Cary, N.C., USA), and GraphPad Prism 7.00software (San Diego, Calif., USA).

Results

Two Lactic Acid Bacteria Strains with the Highest Clearance Ability forUremic Toxin Precursors Were Selected.

To select potential uremic toxin-reducing probiotics, first, a novelscreening platform was established by adding indole or p-cresol tosimulated intestinal juice (pH 7.25) and inoculating it with isolatesfrom Mongolian fermented milk. The 60 isolates were isolated from fiveMongolian fermented milk products (Airag, Tarag, and their mixture) andselected because of their distinct genotyping profiles based onenterobacterial repetitive intergenic consensus polymerase chainreaction (ERIC-PCR) analysis (data not shown). Thirteen of 60 isolates(MFM 12, 14-1, 18, 22, 23, 26-1, 27, 29, 30-2, 30-3, 31, 33, and 40)were selected for further identification due to their higher indoleclearance abilities (Table 1). The identification results from theanalysis of 16S rRNA and housekeeping gene sequences indicated that the13 isolates belonged to three lactic acid bacteria species:Lactiplantibacillus plantarum (six isolates), Companilactobacilluscrustorum (six isolates), and Lacticaseibacillus paracasei (oneisolate). To further select potential strains among the 13 strains, bothindole and p-cresol clearance ability as well as the tolerance ofsimulated gastric and intestinal fluid tests were also determined. Threestrains of L. plantarum (MFM 22, 30-2, and 30-3), one strain of C.crustorum (MFM 14-1), and one strain of L. paracasei (MFM 18) wereselected (Table 2).

TABLE 2 Characteristic Properties of MFM Isolates Gastrointestinaltolerance abilities^(a) (Reduction of surviving cells Clearance ability(%)^(b) Taxonomic attribution Strain represented as Log CFU/mL) Indolep-cresol Lactiplantibacillus MFM 22  6.66 ± 0.25^(a)  9.01 ± 0.40^(ab) 6.24 ± 0.36^(a) plantarum MFM 26-1  5.82 ± 0.40^(bc)  7.26 ± 0.38^(c) 4.39 ± 0.22^(bc) MFM 30-2  5.29 ± 0.26^(cd)  9.70 ± 0.36^(a)  4.82 ±0.33^(b) MFM 30-3  6.10 ± 0.28^(ab)  9.03 ± 0.58^(ab)  5.88 ± 0.26^(a)MFM 33  5.37 ± 0.45^(cd)  8.24 ± 1.41^(bc)  3.97 ± 0.46^(c) MFM 40  4.88± 0.34^(d)  8.73 ± 0.76^(ab)  4.26 ± 0.39^(bc) Companilactobacillus MFM12 7.87 ± 1.49 6.94 ± 0.50 3.64 ± 0.50 crustorum MFM 14-1 7.27 ± 0.936.61 ± 0.72 2.78 ± 0.63 MFM 23 8.88 ± 0.74 7.09 ± 0.14 3.15 ± 0.56 MFM27 8.56 ± 1.35 7.07 ± 0.31 3.07 ± 0.29 MFM 29 8.89 ± 0.78 7.30 ± 0.163.56 + 0.33 MFM 31 8.10 ± 1.62 7.57 ± 0.92 2.51 ± 1.13Lacricaseibacillus MFM 18 7.85 ± 0.30 9.42 ± 0.86 4.31 ± 0.22 paracasei^(a)Gastrointestinal tolerance abilities of MFM isolates after in vitrodigestion in simulated gastric juice and simulated intestinal juice.^(b)Indole and p-cresol clearance ability of MFM isolates after a 48 hincubation in simulated intestinal juice. The results are presented asthe mean ± SD (n = 3). Means in columns with different superscriptletters among the same species are significantly different (p < 0.05).

Additionally, the above selected strains were mixed to compare theirclearance ability with that of the selected single strains (Table 3).The results showed that the probiotics mixed with MFM 30-3 and MFM 18(D3) demonstrated the highest clearance ability of indole (3.32±0.15%)and p-cresol (3.47±0.16%) compared with the other groups. A combinationof two strains (MFM 30-3 and MFM 18) was selected and named lactic acidbacteria mix (Lm) for further animal studies (FIG. 1).

TABLE 3 Various Combinations of Bacterial Strains Single strain Doublestrains Triple strains S1: MFM 22 D1: MFM 22/18 T1: MFM 22/18/14-1 S2:MFM 30-2 D2: MFM 30-2/18 T2: MFM 30-2/18/14-1 S3: MFM 30-3 D3: MFM30-3/18 T3: MFM 30-3/18/14-1 S4: MFM 18 Ratio: 1:1 Ratio: 1:1:1 S5: MFM14-1 Bacterial content: 10⁹ CFU/mL Bacterial content: 10⁹ CFU/mLBacterial content: 10⁹ CFU/mLPretreatment with Lm Prevented the Symptoms of Adenine-Induced RenalInjury in Mice.

In vivo, after 18 days of 0.2% adenine administration, classic kidneylesions, including inflammation and fibrosis in the renal interstitium,atrophy, degeneration, necrosis, regeneration, and hyaline cast in therenal tubule, crystal deposition in the cortical surface, and dilationof Bowen's capsule in the renal corpuscle, were observed in CKD mice,with significantly increased kidney injury scores and fibrosis areascompared with the control group (p<0.05) (FIG. 2A). These findings werefurther confirmed by the activation of the potent fibrogenic factor TGFβand the fibrosis markers fibronectin and collagen 1 (p<0.05) (FIG. 2B).BUN and CRE in serum, which are indicators of renal function, were alsoelevated significantly (p<0.05) (FIG. 2C). However, Lm intervention (LDand HD) prevented damage to the kidney structure by reducing the levelof atrophy, degeneration, hyaline cast, dilation of Bowman's capsule,and crystal deposition, with a significantly lower kidney injury scoreand fibrosis area than their CKD counterparts (p<0.05) (FIG. 2A). Lmtreatment significantly improved tubulointerstitial fibrosis by markedlyreducing the expression of TGFβ (p<0.1) and its downstream activation offibronectin (FIG. 2B). Downregulation of serum BUN and CRE was alsoobserved in the LD group (p<0.1), suggesting a preventive effect of CKDprogression by Lm (FIG. 2C).

Elevated Oxidative Stress and Immunosuppression in CKD Mice WerePartially Restored by Lm Treatment.

The possible mechanisms involved in the preventive effect of Lmtreatment on CKD were then investigated. Systematic oxidative stress andinflammation as well as immune dysregulation were well recognized ascritical exacerbating factors in the progression of CKD. Thus,antioxidant enzymes and inflammation-related cytokines in the kidneywere studied. The results indicated that the CKD group had elevatedoxidative stress; significantly lower GSH, catalase, and GPx levels; andhigher SOD levels than the control group. However, the levels ofcatalase and GPx were significantly restored (p<0.05) by Lm interventionin CKD mice (FIG. 3A). It was also found that the levels ofproinflammatory cytokines (TNF-α and IL-6), the anti-inflammatorycytokine IL-10, and the protein expression of TLR4 in the kidneys of CKDmice were significantly lower than those in the kidneys of control mice(p<0.05) (FIGS. 3B-3C), suggesting immunosuppression in CKD mice. Theexpression of MPO, an enzyme that has a positive correlation with theinflammatory state, was significantly increased in CKD mice comparedwith control mice (p<0.05) (FIG. 3C). In contrast, Lm supplementationrestored the levels of TNF-α and IL-6 and decreased the expression ofMPO (p<0.05) (FIGS. 3B-3C).

Lm Intervention Reduced the Levels of Uremic Toxins and TheirPrecursors.

The indole and p-cresol levels were first determined. The resultsindicated that colonic indole and p-cresol were increased in CKD mice,resulting in significantly increased serial IS and PCS (p<0.05) comparedwith control mice. The low-dose Lm (Lm-LD) intervention showed a trendof reduced p-cresol (p<0.1) and further significantly downregulated thelevels of serum IS and PCS (p<0.05). The high-dose Lm (Lm-HD)intervention also showed a trend of reduced p-cresol (p<0.1) and furthersignificantly downregulated the levels of serum PCS (p<0.05), suggestingthat Lm administration has positive impacts on the improvement of CKDprogression (FIG. 4) by modulating the uremic toxin precursor.

Lm Intervention Improves Intestinal Barrier Integrity.

For intestinal barrier integrity, a significant increase in fluorescenceintensity in the serum of the CKD group was observed (p<0.05) after oraladministration of FITC-dextran. Treatment with Lm-LD significantlyreduced gut permeability (p<0.05) in CKD mice (FIG. 5A). The SCFAresults indicated that although probiotic treatment had no effects onthe elevation of acetic acid and propionic acid, Lm intervention showeda trend of upregulating the level of butyric acid (FIG. 5B).

Lm Intervention Significantly Recovered Gut Dysbiosis and ChangedEnriched Taxa in the Colon of CKD Mice.

The gut microbial composition in the colon was further analyzed toclarify the role of Lm in the modulation of the intestinal microbiota.The alpha diversity indices (Observed_otus, Chao 1 and Shannon) weredecreased significantly in the CKD group, suggesting the transition froma more even and diverse community to a more dominant and identicalcomposition in the intestinal environment, which was driven by CKDinduction. Administration of Lm-LD led to a significant restoration ofrichness and abundance in CKD mice (FIG. 6A). Beta diversity, calculatedby PCA, was utilized to examine microbial composition differencesbetween the tested groups. The PCA plot showed that PCA1 and PCA2explained 27.9 and 12.2% of the variation in gut microbiota composition,respectively. Notably, obvious intergroup distances tended to formdistinct clusters among each group, illustrating the dissimilar gutmicrobiota harbored in the colon (FIG. 6B). These findings suggestedthat the dysbiotic state due to CKD induction could be modulated throughLm intervention toward an intermediate configuration between control andCKD mice.

Since the data indicated that the Lm-LD group demonstrated greaterefficiency than the Lm-HD group in alleviating CKD progression andimproving dysbiosis, taxonomic differences between the CKD and Lm-LDmice were further identified. Administering Lm-LD specificallyinfluenced 10 genera (Faecalibaculum, Coriobacteriaceae UCG 002,Lactococcus, Negativibacillus, Turicibacter, Ruminiclostridium 6,Parasutterella, Eubacterium xylanophilum group, Ruminococcaceae UCG 010and Staphylococcus) and 3 species (Lactococcus lactis, Staphylococcussciuri, and Ligilactobacillus murinus) which were commensal bacteriaespecially short chain fatty acid (SCFA) producers in the gut,indicating that the diminishment of these specific bacteria caused byCKD induction could be restored by Lm supplementation (FIGS. 7A-7C).

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The bacterial strain,probiotic composition comprising the bacterial strain, and uses thereofare representative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. Modificationstherein and other uses will occur to those skilled in the art. Thesemodifications are encompassed within the spirit of the invention and aredefined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitations,which are not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

1. An isolated bacterial strain of Lactiplantibacillus plantarum subsp.plantarum MFM 30-3 deposited under the DSMZ Accession No. DSM
 34213. 2.A probiotic composition comprising an isolated bacterial strain ofLactiplantibacillus plantarum subsp. plantarum MFM 30-3 deposited underthe DSMZ Accession No. DSM 34213, and optionally, one or more additionalprobiotic organisms that enhance the probiotic activity of theLactiplantibacillus plantarum subsp. plantarum MFM 30-3.
 3. Theprobiotic composition of claim 2, which comprises an isolated bacterialstrain of Lactiplantibacillus plantarum subsp. plantarum MFM 30-3deposited under the DSMZ Accession No. DSM 34213, and an isolatedbacterial strain of Lacticaseibacillus paracasei subsp. paracasei MFM 18deposited under the DSMZ Accession No. DSM
 34212. .
 4. The probioticcomposition of claim 2, wherein the composition is in solid, liquid orsemisolid form.
 5. The probiotic composition of claim 2, wherein thecomposition is a food, a food supplement, a nutraceutical, or atherapeutic.
 6. A method for preventing or treating chronic kidneydisease in a subject in need thereof comprising: administering to saidsubject a pharmaceutically effective amount of the composition of claim2.
 7. The method of claim 6, wherein the composition comprises anisolated bacterial strain of Lactiplantibacillus plantarum subsp.plantarum MFM 30-3 deposited under the DSMZ Accession No. DSM 34213, andan isolated bacterial strain of Lacticaseibacillus paracasei subsp.paracasei MFM 18 deposited under the DSMZ Accession No. DSM
 34212. 8.The method of claim 6, which reduces the content of the indicativemolecule selected from indole, p-cresol, indoxyl sulfate, or p-cresylsulfate.
 9. The method of claim 6, which reverses gut dysbiosis andrestores the abundance of commensal bacteria.
 10. The method of claim 6,which improves intestinal barrier integrity.